Mutations in the αGal gene result in the sphingolipidosis named Fabry disease [1]. The enzymatic defect is inherited as an X-linked disorder and is associated with a progressive deposition of the glycosphingolipids, including globotriaosylceramide, galabioasylceramide, and blood group B substance. In affected males this leads to early death due to occlusive disease of the heart, kidney, and brain.
De Duve [2] first suggested that ERT might be a successful approach to the treatment of lysosomal storage defects such as Gaucher's and Fabry disease. For Gaucher's disease, ERT produced unequivocal clinical responses [3, 4] that were subsequently confirmed by others [5-7]. Classical Fabry disease patients lack detectable levels of αGal [1] so it should not be surprising that more than 80% of Fabry patients treated with agalsidase-beta [8] and more than 50% treated with agalsidase-alfa [9] developed an immune response. The antibodies produced are primarily of the IgG class and a fraction of the antibodies appear to exhibit neutralizing properties. These antibodies have been associated with an increase in urinary globotriaosylceramide levels due to the uptake of immune-enzyme complexes by granulocytes in the bloodstream and macrophages in the tissues [10-12].
ERT for Fabry disease patients was initially undertaken for males with the classic form of the disease (no detectable αGal activity) in a variety of clinical trials [8, 9, 13-16], but therapy is now also underway for heterozygous females with Fabry disease [17-19] and is under consideration for children [20-22] and adults with atypical (low levels of enzyme) Fabry disease [23]. The two products used for ERT in Fabry disease patients have been compared [24]. The pattern of glycosylation on αGal has been analyzed [25] and its importance for activity [26] and uptake by cells has been established [27, 28]. The limitations of current approaches for ERT for Fabry disease and the need for improved techniques have been discussed [10, 29, 30]. Efforts for gene therapy for Fabry disease are underway [31-38] and molecular chaperones are under investigation for specific alleles [39-41]. Substrate reduction therapy as an augmentation to ERT has been evaluated [42]. There are several reviews on the general topic of ERT for lysosomal storage diseases [43-47].
Expression of the human αGal has been reported in Escherichia coli [48], baculovirus [49, 50] Chinese hamster ovary cells [51] and human foreskin fibroblasts [52]. The highest levels of heterologous αGal expression were observed in Pichia pastoris [53]. Recombinant αGal has also been produced in a modified strain of Saccharomyces cerevisiae that synthesized glycoprotein lacking the outer chain of N-glycan, a structure that is specific to yeast but not humans [28, 54]. When this αGal was introduced into Fabry patient fibroblasts or a Fabry mouse model, there was hydrolysis of accumulated substrates [28, 54]. The methylotrophic yeast P. pastoris is the most highly developed of a small group of alternative yeast species chosen for their advantages over S. cerevisiae as expression hosts [55, 56]. Two attributes critical in its selection are the existence of well-established fermentation methods and the presence of the tightly regulated methanol-inducible promoter. AOX expression is undetectable by enzyme assay or mRNA production in cells cultured on carbon sources such as glycerol, but constitutes up to 30% of total soluble protein in methanol-grown cells. Heterologous genes under the control of the PAOX1 promoter can be maintained in an expression-off mode on a non-methanolic carbon source in order to minimize expression of potentially toxic heterologous proteins during cell growth. The P. pastoris expression system has now been successfully used to produce a number of heterologous proteins at commercially useful concentrations [57].
Lysosomal enzymes such as αGal are glycoproteins that are modified in the Golgi to contain N- or O-linked carbohydrate structures [58]. The human αGal is glycosylated at Asp residues 139, 193, and 215 [26] with branched carbohydrate structures that vary in composition and sequence depending upon the host species and tissue type [25]. For example, the enzyme purified from humans contains variable amounts (5-15%) of asparagine linked complex and high mannose oligosaccharide chains [1]. Consequently, multiple forms are present in SDS gels and in isoelectric focusing experiments that correspond to the plasma and various tissue forms. The Carboxyl-Terminal Truncations of the Human α-Galactosidase A recombinant human αGal preparations used therapeutically are produced in human and CHO cells and these have distinct glycosylation patterns and differ in levels of sialic acid and mannose-6-phosphate [24]. The recombinant αGal produced in insect cells [49, 50] and in P. pastoris [53] contain variable levels of mostly complex and high mannose side chains, respectively. Glycoproteins produced in P. pastoris typically contain from 6 to 14 mannose units (Man6-GlcNac2 to Man14GlcNac2) that sometimes produces a Gaussian-like distribution of oligomannosides that may center near Man12GlcNac2 to Man13GlcNac2 [59].
These carbohydrate moieties serve a structural and functional role. For example, it has been demonstrated that glycosylation, particularly at Asn-215, is required for enzyme solubility [26]. Also, uptake of the enzyme by cells in vivo is affected by terminal mannose-6-phosphate residues on the enzyme [27], and the 10-12 sialic acid residues on the plasma form of the enzyme accounts for the prolonged circulatory half-life of the enzyme compared to the tissue form with only one or two sialic acid residues [60]. The identification of these multiple forms as derivatives of the same protein in purified enzyme preparations can conveniently be monitored by treatment with specific N-glycosidases or by Western blots.
Fabry disease patients with adverse reactions to the infusions are currently treated with antihistamines and antipyretics and the initial immune response has been manageable to date [61, 62], but it can be anticipated that life-long treatment required for these patients will lead to unacceptable levels of neutralizing antibodies. In this context it is reasonable to devise approaches to circumvent these adverse reactions and the development of derivatives of the enzyme with more activity per mg is a logical approach. Miyamura and coworkers [63] reported that carboxyl-terminal deletions of 2 to 10 amino acids of αGal led to an increase in activity of about 4 to 6-fold as compared to wild type (WT). However, this data was qualitative or semi-quantitative and relied on comparison of the amounts of mRNA present in Northern blots to αGal enzyme activity during transient infection of COS-1 cells. Here we use a P. pastoris expression system for the construction and purification of mutant enzymes with C-terminal deletions. The quantitative results reported here with purified enzymes reveal that C-terminal deletions results in an increase (Δ2, Δ4, Δ6, and Δ10) or decrease (Δ8) in enzyme activity.
Accordingly, there is a need for a method to purify recombinant α-Galactosidase A that provides high yield and maintains enzymatic activity.